Soluble antigen-antibody complexes

ABSTRACT

A composition of matter and method useful for purposes such as identification of spirochetes and electron microscopic localization of tetanus toxin in nervous tissue; the composition being prepared by forming a soluble antigenantibody complex, such as peroxidase-antiperoxidase, by the addition of a moderate antigen excess to immune precipitates and subsequent pH adjustment to 2.3. Upon neutralization, the antigen reequilibriates with the antibody into soluble complexes of homogeneous composition, and the complex is separated from free antigen by precipitation with a predetermined chemical agent, such as ammonium sulfate regarding the aforementioned peroxidaseantiperoxidase complex.

United States Patent [191 Sternberger et al.

[ SOLUBLE ANTIGEN-ANTIBODY COMPLEXES [75] Inventors: Ludwig A.Sternberger, Lutherville,

Md.; John J. Cuculis, Fawn Grove, Pa.; Howard G. Meyer, White Hall, Md.

[73] Assignee: The United States of America as represented by theSecretary of the Army 22 Filed: Apr. 8, 1971 21 Appl. No.2 132,457

[52] U.S. Cl 195/99, 195/68, 195/63, 195/103.5 R [51] Int. Cl Cl2k 1/00[58] Field of Search l95/l03.5 R, 63, 195/68 [56] References Cited OTHERPUBLICATIONS Oncley et al., J. Phys. Chem. 56:90-92 (1952) Singer etal., J. Am. Chem. Soc. 77:3499-3504 1 Nov. 20, 1973 Cothran et al., Ann.Allergy 22:259-268 (1964) Singer et al., J. Am. Chem. Soc. 74:1794-1802(1952) Singer et al., lbid., 77:4851-4857 (1955) Primary ExaminerAlvinE. Tanenholtz Assistant ExaminerMax D. Hensley Attorney-Harry M.Saragovitz, Edward J. Kelly, Herbert Her] and Bernard J. Ohlendorf 57ABSTRACT A composition of matter and method useful for purposes such asidentification of spirochetes and electron microscopic localization oftetanus toxin in nervous tissue; the composition being prepared byforming a soluble antigenantibody complex, such asperoxidase-antiperoxidase, by the addition of a moderate antigen excessto immune precipitates and subsequent pH adjustment to 2.3. Uponneutralization, the antigen reequilibriates with the antibody intosoluble complexes of homogeneous composition, and the complex isseparated from free antigen by precipitation with a predeterminedchemical agent, such as ammonium sulfate regarding the aforementionedperoxidaseantiperoxidase complex.

10 Claims, 1 Drawing Figure PAIENIEnuuvzo 1975 3.773.625

lNl/ENTORS Ludwig A. Star/merger John J. Cucu/is Howard 6. Meyer 24 7%.5

BY W M 1 SOLUBLE ANTIGEN-ANTIBODY COMPLEXES DEDICATORY CLAUSE Theinvention described herein may be manufactured, used, and licensed by orfor the Government for governmental purposes without the payment to usof any royalty thereon.

Our invention relates to a new unlabeled antibody enzyme method ofimmunohistochemistry and a new soluble antigen-antibody complexcomposition of matter employed therein.

Our new method and composition of matter has utility, among other uses,in the identification of spirochetes and the electron microscopiclocalization of tetanus toxin in nervous tissue.

The prior art method for preparing soluble antigenantibody complexesconsisted of precipitating the antibody with specific antigen andredissolving the washed precipitate by shaking with excess antigen, asdescribed by S. J. Singer and D. H. Campbell in Physical ChemicalStudies of Soluble Antigen Antibody Complexes. 1. The Valence ofPrecipitating Rabbit Antibody, J. Amer. Chem. Soc. 7421794, 1952 and inPhysical Chemical Studies of Soluble Antigen Antibody Complexes. V.Theromodynamics of the Reaction Between Ovalbumin and Its RabbitAntibody, J. Amer. Chem. Soc. 7714851, 1955. The aforementioned priorart method presented a multiplicity of problems which include theresolubilization of the antibody with specific antigen from theprecipitate being incomplete unless exceedingly large amounts of antigenwere employed; the shaking of the antigen-antibody precipitate withexcess antigen being a prolonged procedure which resulted in partialdenaturation of the precipitate constituents; and the soluble complexesbeing heterogeneous mixtures which included free antigen, complexes ofantigen-antibody of ratio of 2:1, complexes of antigen-antibody of ratioof 3:2, and a mixture of unidentified antigen-antibody complexes oflower ratios. Our invention was conceived and reduced to practice tosolve the aforementioned problems and to satisfy the long felt need fora soluble antigen-antibody complex for use in immunohistochemistry whichis homogeneous as to composition, molecular weight, and molecular sizeand which does not require a large excess of antigen to form and doesnot result in denaturation of the complex constituents during theformation of the complex.

The principal object of our invention is to provide a solubleantigenantibody complex and method of immunohistochemistry which iseconomical and rapid to use and does not result in denaturation of anycomplex constituents during the complex formation.

Another object of our invention is to provide a soluble antigen-antibodycomplex and method of immunohistochemistry wherein the antigen isreequilibriated with the whole of the immune precipitate rather thanonly with that fraction of the precipitate that is slowly redissolved byexcess antigen.

A further object of our invention is to provide a solubleantigen-antibody complex and method of immunohistochemistry wherein themost avid antibodies are employed which engender the lowest chance ofdissociation from tissue.

Other objects of our invention will be obvious or will appear in thespecification hereinafter set forth.

The drawing is a diagram of a molecule of peroxidase-antiperoxidasecomplex, hereinafter referenced as PAP and hereinafter described. In thedrawing, PO designates a peroxidase antigen, Fab designates an antibodyfragment containing a specific combining site, Fc designates acrystalizable and class determining antibody fragment site, and 8-8designates a bisulfide linkage; the antibody being a material such asimmunoglobulin G and hereinafter referenced as lgG.

Generally, our invention is described as a soluble antigen-antibodycomplex prepared by the addition of a moderate antigen excess to immuneprecipitates" and subsequent pH adjustment to 2.3. Upon neutralization,the antigen, such as peroxidase and hereinafter refer enced as PO,reequilibrated with the antobody into soluble complexes of homogeneouscomposition, and the complex was separated from the free antigen byprecipitation with a predetermined chemical agent, such as ammoniumsulfate regarding the peroxidaseantiperoxidase complex subsequentlydescribed.

Our invention will now be described in detail as follows.

EXAMPLE I Fourteen milliliters of an aqueous solution containing 950ugPO/ml, such as Horseradish Peroxidase, were mixed with 40.0 ml rabbitantiserum to PO, and the resulting immune precipitate was allowed todevelop at room temperature for 1 hour. Following precipitation, thefollowing procedure was carried out in refrigerated containers atapproximately 0C to 2C or in a cold room at approximately 5C. Theprecipitate was centrifuged at 10,000 rpm for about 20 minutes by aconventional centrifuge and washed three times with 500 ml portions ofchilled saline, 0.15 N NaCl. The precipitate was then resuspended in27.0 ml of an aqueous solution containing 1,900ug PO/ml and dissolved byadjustment of the solution pH to 2.3 at 1C. Two drops of 1.0 Nhydrochloric acid and 12 drops of 0.1 N hydrochloric acid were consumedin the pH adjustment. The solution was immediately neutralized toapproximately pH 7.4 and 2.7 m1 of 0.08 N sodium acetate solution and0.15 N ammonium acetate solution were added to the neutralized solution.An equal volume of an ammomium sulfate solution, saturated atapproximately 2C to 5C, was slowly admixed to the foregoing solution.The resulting suspension was stirred for about 25 minutes andcentrigufed for about 16 minutes at 17,500 rpm by a conventionalcentrifuge to remove the suspended particles from the neutralizedsolution, and the supernatant liquid was retained for further analysis.The precipitate of the suspended particles was washed once in 0.5saturated ammonium sulfate solution, dissolved in 30 ml of water anddialyzed against three changes of 15 liters each of sodium-ammoniumacetate saline (13.5 liters saline, such as 0.15 N NaCl, 1.5 literswater, ml 1.5 N sodium acetate and 75 ji 3 N ammonium acetate solution).At least one day was allowed for each change of dialysis. A smallprecipitate that had formed after dialysis was conventionally removed bycentrifugation at 17,500 rpm for about 16 minutes. Five 2.0 ml portionsof the resulting solution were frozen with Dry Ice and acetone andstored at 20C for future use. Optionally, the resulting solution can bestored in a cold room at approximately 2C to 5C.

As described in the above example for preparing PAP, our procedure canbe applied to the preparation of soluble complexes from anyprecipitating antigenantibody system, and theprocedure can be carriedout at any temperature, including room temperature. The excess ofantigen may be any amount higher than 3 times that necessary for theoriginal precipitation of antibody, and the PH of dissociation may beanywhere above pH 9.0 or below 5.0.

When treated with the above prepared PAP by conventional stainingtechnique, spirochetes obtained on the fourteenth day after inoculationof cortisonetreated rabbits became greatly thickened and deeply stainedwhen treated with the unlabeled antibody peroxidase, Example IIprocedure below. The organisms were clearly visible at l-fold and550-fold bright field light microscopic magnification. When the stainingprocedure dilutions of normal serum were substituted for dilutions ofimmune serum or when the rabbit antiserum was entirely omitted, theorganisms were only faintly stained. Nevertheless, they were visible at550-fold bright field magnification. Had staining been entirelynegative, the organisms should have been invisible at this or higherfield light microscopic magnification, because the diameter of T.pallidum is only 0.1 It is considered that the low degree of staining inthe control preparations, particularly in those in which rabbitantiserum was entirely omitted, was due to small amounts of antibodiesformed in the host rabbits within 14 days of infection despiteimmunosuppressive treatment by cortisone. Although the organisms may nothave been combined with antibody in the lesion itself, theyunquestionably have had contact with host serum during isolation. Toexclude or minimize this possibility, other spirochetes were obtained onthe sixth day after inoculation. When stained according to Example IIbelow, and after exposure to dilutions of antisyphilitic serum, thespirochetes became as intensely stained as the organisms obtained on thefourteenth day described above. In controls stained after omission ofrabbit serum and with the otherwise complete staining procedure, theorganisms were fainter than those obtained on the aforementionedfourteenth day. The suspensions of organisms obtained on the sixth daywere not as heavy as those obtained on the fourteenth day, and it wasconcluded that small amounts of antibody indeed had formed in the seraof cortisone-treated rabbits 14 days after infection; but on the sixthday only minimal amounts of antibody were available. Spirochetesobtainedon the sixth day after infection and stained by procddure of Example I]below after exposure to dilutions of normal serum, instead ofantisyphilitic serum, also were less marked than similarly treatedorganisms obtained on the fourteenth day, but, nevertheless, they werevisible. Since normal animals contain saprophytic spirochetes and sincethese spirochetes cross-react with T. pallidum, attempts were made toblock these crossreacting antigenic sites. The blocking was accomplishedby preapplication of normal sheep serum, Example III below, on the basisthat normal sheep contain antibodies against saprophytic spirochetessimilar to those of normal rabbits and that any sheep antispirochetalantibody combined with the organisms would not react with thesubsequently applied sheep antirabbit IgG. When Example III procedurewas employed with various'dilutions of rabbit antisyphilitic sera on.spirochetes obtained 6 days after infection, the sensitivity was notimpaired relative to Example 11 procedure. However, controls treatedwith dilutions of normal rabbit serum instead of antisyphilitic serumwere fainter than those stained by Example II procedure. Most of theorganisms remained invisible and the rare ones seen were only faintlyvisualized and had to be searched for on the slide. This contrasts withthe prominence and abundance of the organisms in any field of the slideupon specific staining. When rabbit serum was omitted in the otherwisecomplete staining Example III procedure, the spirochetes remainedentirely invisible in bright field microscopy. Similarly, when in theotherwise complete staining procedure (using any dilution ofantisyphilitic or normal rabbit serum) and anti-lgG being omitted, thespirochetes were not visualized.

We considered reactions positive only when the spirochetes were deeplystained, greatly thickened and immediately apparent on low power(l25-fold) and medium powerr (550-fold) bright field microscopy, and theend points of titers of antiserawere estimated by this criterion. Theend point is obvious even without comparison with controls. It isconsidered that positive reactions can be identified within the skill ofthe art at higher titers is comparison with controls are made.

A comparison of unlabeled antibody peroxidase titers and indirectfluorescent antibody titers obtained after application of rabbit serashowed that the unlabeled antibody peroxidase method was about to 1,000times more sensitive in the evaluation of the five strongly reactingantisyphilitic sera. See Table I below. On the other hand, the reactionof normal sera by immunoflorescence was not exacerbated with theunlabeled antibody peroxidase technique. Hence, the method was also 100to 1,000 times more specific. For reasons unknown, the titers of the twopoorly reacting postinoculation sera were similar with theimmunofiuorescence and the unlabeled antibody peroxidase techniques.

In the staining experiments described above, only the rabbit sera werediluted, and the other three staining factors used prior to osmicationwere as follows: (a) anti-immunoglobulin G, hereinafter referenced aslgG, undiluted; (b) PAP, concentrations given in Table II below; and (c)3,3-diaminobenzidene tetrahydrochloride hereinafter referenced as DAB,0.05%; hydrogen peroxide 0.01%. However, titers remained unaffected whenany one or two of factors (a), (b), or (c) above were diluted 1:10 inaddition to the usual dilutions of rabbit sera. When all three of theaforementioned factors were diluted, staining intensity was diminished.

Our inventive method has also been specific at high sensitivity for theelectron microscopic localization of tetanus toxin in nervous tissue.However, it was necessary to apply the staining materials prior toembedding for electron microscopy using paraformaldehyde-fixed tissue,sectioned in the frozen state at 20 p. thickness.

Analysis of the supernatant liquid from the ammonium sulfateprecipitation, referenced in the above Example I, in the preparation ofPAP revealed no significant amount of anti-PO. See Table II below. Whentrace amounts of anti-PO present are combined with P0 as complexes of 2molecules of PO and 1 molecule of anti-PO, they account for only aminimal fraction of either the free P0 in the supernatant liquid or ofthe P0 in the PAP precipitated with the ammonium sulfate. Less thanone-third of the PO used for preparation of PAP appeared as free P0 inthe ammonium sulfate supematants which indicated that most of the excessP0 was recovered in the complex with high efficiency.

TABLE I Comparison of Sensitivities of the Unlabeled Antibody Peroxidaseand the Fluorescent Antibody Methods in the Detection of Spirocheteswith Dilutions of Immune Sera Unlabeled Antibody Peroxidase FluorescentAntibody Method Method Rabbit Pre- Post- Pre- Post No.inocuinocuinocuinoculation lation lation lation titer" titer titer titera. Example II procedure.

b. Reciprocals of dilution c. Not done.

TABLE II Composition of Ammonium Sulfate Supematants Remaining in thePreparation of Soluble Peroxidase-Antiperoxidase Complex (PAP) byAcid-Dissociative Exchange of IMMUNE Precipitates with Excess AntigenPrepa- Anti- Free Free ration Volume P P0 P0 P0" ml [Lg/ml Lg/ml ug/mlTotal A 23 176 42 156 26 B 34 357 25 344 30 C 405 39 385 D 54 123 0 12322 E 50 3.05 44 283 33 a. Based on each anti-PO molecule binding 2molecules of PO b. (Free P0 in supernatant 100/(T0tal P0 in supernatant)(P0 in PAP).

EXAMPLE II Immunohistochemical staining was carried out as follows. Oneto two drops of reagents were applied in sequential steps to spirocheteson glass slides; the slides having been placed on moistened beaker matsin covered Petri dishes. After each step, the slides were first rinsedwith 10 ml of wash solution slowly dripping from a pipette and thenwashed for minutes by immersion in wash solution. The spirochetes on theslides were than sequentially treated according to the following steps.

Step 1: Dilutions of rabbit antisyphilitic serum for 30 minutes followedby rinse and wash in tris 0.15 N NaCl, hereinafter referenced as T(serum was diluted in T containing 0.1 percent gelatin).

Step 2: Sheep anti-rabbit IgG for 30 minutes followed by rinse and washin T.

Step 3: PAP for 30 minutes followed by rinse in T and wash in triswater, hereinafter referenced as TAD.

Step 4: A freshly prepared solution containing 0.05% of3,3-diaminobenzidene tetrahydrochloride, hereinafter referenced as DAB,and 0.01% of hydrogen peroxide for about 5 minutes followed by rinse andwash in water.

Step 5: The slides were placed in a glass chamber containing osmiumtetroxide crystals; the chamber was sealed with Teflon tape; the chamberwas placed upon a water bath at about C for about 30. minutes; andstaining was accomplished after the slides were removed from the chamberaccording to the procedure reported by Seligman, A.M., Wasserkrug, EL.and I-Ianker, J.S.: A New Staining Method (0T0) for Enhancing Contrastof Lipid Containing Membranes and Droplets in Osmium-Fixed Tissue withOsmiophilic Thiocarbohydrazide (TCH). J. Cell Biol. 30:424, 1966.

EXAMPLE In Same as Example II above except that the slides were treatedwith normal sheep serum for about 30 minutes followed by rinse and washwith T prior to step 1 of Exampl llt Staining with fluorescentantibodies was done by the indirect method reported by Deacon, U.E.,Lucas, J .B., and Price, E.V.: Fluorescent Treponemal Antibody-Absorption (F TA-AB) Test for Syphilis. J .A.M.A. 198:624, 1966.

Irrespective of the serum pool used, the ratio of PC to anti-PO in eachpreparation of PAP was close to 3:2. See Table III below. On doublediffusion, rabbit serum (anti-PO) and anti-rabbit IgG formed seven linesof precipitation. The major line, due to IgG, exhibited identity withthe line formed by PAP and anti-IgG. The

single line formed between PAP and anti-PO spurred on the IgG linebetween rabbit anti-PO and anti-rabbit IgG. The spur does not in itselfindicate identity of PAP reactive with anti-PO and anti-IgG, becauseanti-PO cannot diffuse into the domain surrounding the anti- IgG welland would yield spur formation even if PO and anti-IgG in PAP wereseparate components. However, addition of anti-IgG to PAP precipitated99.1% of enzymatic activity, which attested to the fact that the P0 inPAP was indeed bound and that there was no significant amount of freePO. Comparison of diffusion rates of PAP and IgG against anti-IgG showedthat PAP diffusion was retarded relative to that of IgG, which indicatedthat the IgG was bound in PAP inasmuch as any significant amount of freeIgG would have formed faster moving bands and would have precluded theappearance of the trailing PAP band. Furthermore, PAP gave a single peakon sedimentation and a regular appearance on negative as well aspositive staining in electron microscopy; all of which attest to thehomogeneity of the complex.

On electrophoresis, PO moved slower toward the anode than IgG, and PAPmoved faster toweard the anote than either P0 or IgG. This cannot be dueto neutralization of charge upon reaction of P0 with IgG, because, insuch case, the mobility would have been intermediate. The reason forthis acceleration in movement toward the anode is considered to be dueto the larger molecular size of PAP in comparison to PO and IgG, whichresults in retardation relative to the buffer in the agar gel sieve andwhich attests again to the fact that PO and IgG are bound in PAP evenwhen in an electric field. The single band formed by PAP with anti-POextended farther toward the cathode than the band formed with anti-lgG.This shows that the distribution of charge on the complex isheterogeneous and that in some forms of complex antigenic determinantsof IgG predominate and in other forms of complex antigenic determinantsof PO predominate. Similarly, the degree of impairment of enzymaticactivity as a result of the reaction of P0 with anti-PO in the formationof PAP was heterogeneous, ranging from 2 to 62. See Table III below. Theenzymatic activity of PO alone was not af fected by treatment at pH 2.3,1C.

The PAP complex consisted of 3 molecules of PO for each 2 molecules ofanti-PO which suggested a molecular weight of 432,000 or any multiplethereof for PAP. The dedimentation constant of PAP was determined to be11.98 X and the diffusion constant of 2.48 X 10* was determined byanalysis; yielding a molecular weight of 429,000 by sedimentationvelocity. Sedimentation equilibrium gave a molecular weight of 410,000by an independent measurement. Using this figure and the sedimentationconstant, the calculated diffusion constant becomes 3.59 X 10".

These data indicate that PAP is a pentameric molecule, as shown in thedrawing, consisting of two IgG and three PO subunits. This structure wasconfirmed by direct electron microscopic observation after negative orpositive staining. PAP wassumed the form of pentagonal rings withdiameters of 205 A. Since electron micrography gave no evidence oftwisted forms, the rings are considered to be of flat configuration.

alibi fin" of peroxidase and two molecules of anti-peroxidase; saidsubstance having a molecular weight of 429,000 by sedimentationvelocity, a sedimentation constant of 11.98 X 10", and a diffusionconstant of 2.48 X 10".

4. The composition of claim 2 wherein the substance has a molecularconfiguration of flat pentagonal rings having diameters of 205 A.

5. The composition of claim 3 wherein each side of the pentamericmolecule is 120 A, each peroxidase molecule has a short axis of A and along axis of 56 -A, the specific combining sites of the anti-peroxidasemolecules are joined by a linkage at 108, and a side of thecrystallizable and class determining fragment of the anti-peroxidasemolecules is A.

6. The composition of claim 5 wherein the linkage is a bisulfidelinkage.

7. A rapid method of preparing a specifically purified and solubleperoxidase-antiperoxidase complex for high sensitivity in thehistochemical localization of anti gen comprising the steps of forming asolution containing peroxidase; adding an antibody material to the solu-Composition of soluble peroxidase antiperoxidase complex (PAP) purifiedfrom various pools of antiperoxidase (Anti-PO) sera Antiserum sourcePreparation Serum pool No. Volume used, ml. Anti-PO content, gJml.

Purified peroxidase-antiperoxidase complex Vol. PAP P0 Anti-PO EnzymaticExcess PO obtained, contents, contents, Mole ratio activity, Yield,Preparation employed ml. gJml. [LgJlTll- PO/anti-PO percent percentRatio of total PO used for preparation of PAP to PO employed inprecipitating anti-PO from antiserum at equivalence.

b Taking themmolecular weight of anti-PO as 156,000 and that of P0 as39,800. f lgilrgzymatic activity per mole of P0 in PAP)X1U0 (Enzymaticactivity per mole of PO used for preparation 0 d (Anti-PO in total PAP)XIOO/Antl-PO in total serum pool.

The above description clearly demonstrates that our inventive unlabeledantibody immunohistochemistry method provides high sensitivity in thehistochemical localization of antigen; a simple and rapid method for thepreparation of specifically purified and soluble antigen-antibodycomplex which circumvents the need for the preparation of specificallypurified antibody; and a product which is yielded in high concentration,stability, and homogeneity.

It is obvious that other modifications can be made of our invention, andwe desire to be limited only by the scope of the appended claims.

We claim:

1. A discrete complex composition of matter for use in identifyinginfections and toxin materials, said composition consisting essentiallyof a soluble peroxidaseantiperoxidase complex which is homogeneous incomposition in which the peroxidase-antiperoxidase ratio is threeperoxidase molecules to two anti-peroxidase molecules.

2. The composition of claim 1 wherein the substance is horseradishperoxdose-antihorseradish peroxidase.

3. The composition of claim 1 wherein the substance is a pentamericmolecule consisting of three molecules tion to produce an immuneprecipitate; suspending the immune precipitate in a second solutioncontaining a moderate excess of peroxidase; adjusting the pH of theimmune precipitate suspension to 2.3 at 1C; neutralizing the adjusted pHsuspension; separating the peroxidase-antiperoxidase complex from freeperoxidase in the neutralized suspension by adding a predeterminedchemical agent to the neutralized suspension to effect precipitation ofthe peroxidase-antiperoxidase complex; and finally treating theperoxidase-antiperoxidase complex by the disolution, dialyzing, andseparation of any precipitated impurity.

8. A rapid method of preparing a specifically purified and solubleperoxidase-antiperoxidase complex for high sensitivity in thehistochemical localization of antigen comprising the steps of dissolvingapproximately 950 ug of a peroxidase per ml in an aqueous solutionmedium, mixing about fourteen milliliters of the peroxidase solutionwith about forty milliliters of rabbit antiserum to form an immuneprecipitate, developing the immune precipitate for about one hour atroom temperature, removing the immune precipitate from the aqueousmedium, washing the immune precipitate in a saline solution, suspendingthe washed precipitate in about 27 ml of an aqueous solution containingabout l,900;.tg of a peroxidase per ml, adjusting the pH of the aqueoussolution to 2.3 at 1C to dissolve the suspended precipitate in theaqueous solution, neutralizing the adjusted pH solution to a pH ofapproximately 7.4, adding about 2.7 ml of 0.08 N sodium acetate and 0.15N ammonium acetate to the neutralized solution, adding an equal volumeof a saturated ammonium sulfate solution to the neutralized solutioncontaining the sodium and ammonium acetate to produce a suspension,stirring the ammonium sulfate produced suspension for about 25 minutes,removing the sulfate produced suspended particles from the suspension medium, washing the removed sulfate produced suspended particles in a onehalf saturated ammonium sulfate solution, dissolving the washed sulfateproduced suspended particles in about 30 ml of water, dialyzing thesolution of the ammonium sulfate produced suspended particles againstabout liters of a saline solu tion, removing any precipitate formed, andrecovering the resulting solution.

9. The method of claim 8 wherein the peroxidase is horseradishperoxidase; the immune precipitate is removed by centrifuging at about10,000 rpm for about twenty minutes; the saline solution for washing theimmune precipitate is 0.15 N NaCl and three washings of 500 ml portionseach of the saline solution are used; the pH adjustment to 2.3 at 1 C isaccomplished by the addition of about two drops of 1.0 N HCL and about12 drops of 0.1 N BC]; the saturated ammonium sulfate solution issaturated at approximately 2C to 5C; three dialysis changes are usedwith at least one day allowed for each dialysis change; and the dialysissaline solution comprises about 13.5 liters of 0.15 N NaCl, 1.5 litersof H 0, ml of 1.5 N sodium acetate, and 75 ml of 3N ammonium acetate;and any precipitate after dialysis is removed by centrifuging at about17,500 rpm for about sixteen minutes.

10. The method of claim 8 wherein the solution after dialysis andprecipitate removal is frozen in 2.0 ml portions with dry ice andacetone and stored at 20C for future use.

2. The composition of claim 1 wherein the substance is horseradishperoxdose-antihorseradish peroxidase.
 3. The composition of claim 1wherein the substance is a pentameric molecule consisting of threemolecules of peroxidase and two molecules of anti-peroxidase; saidsubstance having a molecular weight of 429,000 by sedimentationvelocity, a sedimentation constant of 11.98 X 10 13, and a diffusionconstant of 2.48 X 10
 7. 4. The composition of claim 2 wherein thesubstance has a molecular configuration of flat pentagonal rings havingdiameters of 205 A.
 5. The composition of claim 3 wherein each side ofthe pentameric molecule is 120 A, each peroxidase molecule has a shortaxis of 35 A and a long axis of 56 A, the specific combining sites ofthe anti-peroxidase molecules are joined by a linkage at 108*, and aside of the crystallizable and class determining fragment of theanti-peroxidase molecules is 40 A.
 6. The composition of claim 5 whereinthe linkage is a bisulfide linkage.
 7. A rapid method of preparing aspecifically purified and soluble peroxidase-antiperoxidase complex forhigh sensitivity in the histochemical localization of antigen comprisingthe steps of forming a solution containing peroxidase; adding anantibody material to the solution to produce an immune precipitate;suspending the immune precipitate in a second solution containing amoderate excess of peroxidase; adjusting the pH of the immuneprecipitate suspension to 2.3 at 1*C; neutralizing the adjusted pHsuspension; separating the peroxidase-antiperoxidase complex from freeperoxidase in the neutralized suspension by adding a predeterminedchemical agent to the neutralized suspension to effect precipitation ofthe peroxidase-antiperoxidase complex; and finally treating theperoxidase-antiperoxidase complex by the disolution, dialyzing, andseparation of any precipitated impurity.
 8. A rapid method of preparinga specifically purified and soluble peroxidase-antiperoxidase complexfor high sensitivity in the histochemical localization of antigencomprising the steps of dissolving approximately 950 Mu g of aperoxidase per ml in an aqueous solution medium, mixing about fourteenmilliliters of the peroxidase solution with about forty milliliters ofrabbit antiserum to form an immune precipitate, developing the immuneprecipitate for about one hour at room temperature, removing the immuneprecipitate from the aqueous medium, washing the immune precipitate in asaline solution, suspending the washed precipitate in about 27 ml of anaqueous solution containing about 1,900 Mu g of a peroxidase per ml,adjusting the pH of the aqueous solution to 2.3 at 1*C to dissolve thesuspended precipitate in the aqueous solution, neutralizing the adjusteDpH solution to a pH of approximately 7.4, adding about 2.7 ml of 0.08 Nsodium acetate and 0.15 N ammonium acetate to the neutralized solution,adding an equal volume of a saturated ammonium sulfate solution to theneutralized solution containing the sodium and ammonium acetate toproduce a suspension, stirring the ammonium sulfate produced suspensionfor about 25 minutes, removing the sulfate produced suspended particlesfrom the suspension medium, washing the removed sulfate producedsuspended particles in a one half saturated ammonium sulfate solution,dissolving the washed sulfate produced suspended particles in about 30ml of water, dialyzing the solution of the ammonium sulfate producedsuspended particles against about 15 liters of a saline solution,removing any precipitate formed, and recovering the resulting solution.9. The method of claim 8 wherein the peroxidase is horseradishperoxidase; the immune precipitate is removed by centrifuging at about10,000 rpm for about twenty minutes; the saline solution for washing theimmune precipitate is 0.15 N NaCl and three washings of 500 ml portionseach of the saline solution are used; the pH adjustment to 2.3 at 1* Cis accomplished by the addition of about two drops of 1.0 N HCL andabout 12 drops of 0.1 N HCl; the saturated ammonium sulfate solution issaturated at approximately 2*C to 5*C; three dialysis changes are usedwith at least one day allowed for each dialysis change; and the dialysissaline solution comprises about 13.5 liters of 0.15 N NaCl, 1.5 litersof H2O, 75 ml of 1.5 N sodium acetate, and 75 ml of 3N ammonium acetate;and any precipitate after dialysis is removed by centrifuging at about17,500 rpm for about sixteen minutes.
 10. The method of claim 8 whereinthe solution after dialysis and precipitate removal is frozen in 2.0 mlportions with dry ice and acetone and stored at -20*C for future use.